US8663933B2

US8663933B2 - Recombinant protein capable of binding specifically and quickly to troponin I derived from human myocardium

Info

Publication number: US8663933B2
Application number: US13/680,925
Authority: US
Inventors: Atsushi Fukunaga
Original assignee: Panasonic Corp
Current assignee: PHC Corp
Priority date: 2011-12-16
Filing date: 2012-11-19
Publication date: 2014-03-04
Anticipated expiration: 2032-03-13
Also published as: US20130158237A1

Abstract

Provided is a recombination protein which binds specifically to troponin I derived from human myocardium. The recombinant protein includes a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63; and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Application No. PCT/JP2012/001726, with an international filing date of Mar. 13, 2012, which claims priority of Japanese Patent Application No. 2011-275413, filed on Dec. 16, 2011, the entire contents of each of which are incorporated herein by reference.

The Sequence listing in “SEQUENCE LISTING.TXT” created on Feb. 8, 2013 and being 21.0 KB in size is incorporated by reference and is identical to the sequence information in the instant application.

FIELD

The technical field relates to a recombinant protein capable of binding specifically and quickly to troponin I derived from human myocardium.

BACKGROUND

Non Patent Literature 1 and Non Patent Literature 2 disclose that a concentration of troponin I derived from myocardial tissue increases rapidly in the blood of a patient who has suffered acute myocardial infarction.

CITATION LIST Non Patent Literature

Non Patent Literature 1

Aleksei G. Katrukha et. al., “Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex”, Clinical Chemistry, Vol. 43, Issue 8, p.p. 1379-1385 (1997)

Non Patent Literature 2

Till Keller et. al., “Sensitive Troponin I Assay in Early Diagnosis of Acute Myocardinal Infarction”, The NEW ENGLAND JOURNAL of MEDICINE, Vol. 361, pages 868-877 (2009)

SUMMARY

One non-limiting and exemplary embodiment provides a recombinant protein capable of binding specifically and quickly to troponin I derived from human myocardium.

Additional benefits and advantages of the disclosed embodiments will be apparent from the specification and Figures. The benefits and/or advantages may be individually provided by the various embodiments and features of the specification and drawings disclosure, and need not all be provided in order to obtain one or more of the same.

In one general aspect, the techniques disclosed here feature a recombinant protein which binds specifically to troponin I derived from human myocardium. The recombinant protein includes a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63, and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65.

The present disclosure provides a recombinant protein capable of binding specifically and quickly to troponin I derived from human myocardium.

The recombinant protein and the method of the present disclosure can be used for early detection of acute myocardial infarction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an antibody.

FIG. 2 shows a PCR method in the step (c-2).

DETAILED DESCRIPTION

The embodiment of the present disclosure is described below.

(Explanation of Terms)

First, the terms used in the present specification are described.

FIG. 1 shows an antibody. The antibody 1 has a letter “Y” shape. The antibody 1 has two Fab regions and one Fc region. The antibody 1 consists of two heavy chains 2 and two light chains 3. Each heavy chain 2 consists of a heavy chain constant region 1 (22), a heavy chain constant region 2 (23), a heavy chain constant region 3 (24) and a heavy chain variable region 21. Each light chain 3 consists of a light chain variable region 31 and a light chain constant region 32.

Each Fab region consists of the one heavy chain variable region 21, the one heavy chain constant region 1 (22), the one light chain variable region 31, and the one light chain constant region 32. The light chain 3 is connected to the heavy chain 2 through a linker 4. The one heavy variable region 21 is present at the end of the heavy chain 2. The one light chain variable region 31 is present at the end of the light chain 3. An antigen is specifically bound to the antibody 1. In more detail, the antigen is bound specifically to the Fv region, which consists of the heavy chain variable region 21 and the light chain variable region 31. In the present specification, the antigen is troponin I derived from human myocardium.

The recombination protein according to the embodiment includes the light chain variable region 31 consisting of the amino acid sequence represented by SEQ ID NO: 63 and the heavy chain variable region 21 consists of the amino acid sequence represented by SEQ ID NO: 65. The recombinant protein of the present disclosure binds specifically and quickly to the troponin I derived from human myocardium.

The recombinant protein of the present disclosure may be either an antibody or an antibody fragment.

The antibody has a shape of the form of the letter “Y” shown in FIG. 1. The light chain variable region 31 and the heavy chain variable region 21, both of which is included in the antibody of the present disclosure, consist of amino acid sequences represented by SEQ ID NO: 63 and SEQ ID NO: 65, respectively. In the antibody, the light chain variable region 31 is connected to the heavy chain variable region 21 through a linker (not shown).

Examples of the antibody fragment include a Fab antibody fragment, a F(ab′)₂antibody fragment and an scFv antibody fragment.

The Fab antibody fragment consists of one Fab region. In other words, the Fab antibody fragment consists of the one light chain variable region 31 (SEQ ID NO: 63), the one heavy chain variable region 21 (SEQ ID NO: 65), the one light chain constant region 32, the one heavy chain constant region 1 (22), and the linker 4. The light chain constant region 32 is connected to the heavy chain constant region 1 (22) through the linker 4.

The F(ab′)₂antibody fragment consists of two Fab regions. As above, each Fab region consists of the one light chain variable region 31 (SEQ ID NO: 63), the one heavy chain variable region 21 (SEQ ID NO: 65), the one light chain constant region 32, the one heavy chain constant region 1 (22), and the linker 4. These two Fab regions are connected to each other through another linker (not shown). For example, one heavy chain constant region 1 (22) is connected to the other heavy chain constant region 1 (22) through another linker (not shown).

The scFv antibody fragment consists of the light chain variable region 31 (SEQ ID NO: 63), the heavy chain variable region 21 (SEQ ID NO: 65), and a linker. The light chain variable region 31 (SEQ ID NO: 63) is connected to the heavy chain variable region 21 (SEQ ID NO: 65) through a linker (not shown).

As long as the recombination protein is capable of binding specifically and quickly to troponin I derived from human myocardium, the linker connecting the light chain variable region 31 (SEQ ID NO: 63) and the heavy chain variable region 21 (SEQ ID NO: 65) is not specifically limited. An example of the linker is a peptide consisting of 5-20 amino acids. For example, the linker is a peptide consisting of the amino acid sequence represented by GGGGSGGGGSGGGGS (SEQ ID NO: 64). Another example of the linker is a disulfide bond (-sulfur atom (S)—sulfur atom (S)—).

As long as the recombination protein is capable of binding specifically and quickly to troponin I derived from human myocardium, the N-terminal of the light chain variable region 31 (SEQ ID NO: 63) may be modified with an amino acid sequence. The C-terminal thereof may be also modified.

As long as the recombination protein is capable of binding specifically and quickly to troponin I derived from human myocardium, the N-terminal of the heavy chain variable region 21 (SEQ ID NO: 65) may be modified with an amino acid sequence. The C-terminal thereof may be also modified. An example of the amino acid sequence to modify the C-terminal of the heavy chain variable region 21 (SEQ ID NO: 65) is AAALEHHHHHH (SEQ ID NO: 66).

When the recombinant protein of the present disclosure is brought into contact with troponin I derived from human myocardium, the recombinant protein of the present disclosure binds specifically and quickly to the troponin I derived from human myocardium. For example, when the recombinant protein of the present disclosure is mixed with troponin I derived from human myocardium, the recombinant protein of the present disclosure binds specifically and quickly to the troponin I derived from human myocardium.

Detection of the binding of the recombinant protein of the present disclosure to the troponin I derived from human myocardium can be carried out by methods for detecting antigen-antibody binding which are well known to those skilled in the art. Examples of such methods include the ELISA sandwich method.

The recombinant protein of the present disclosure can be produced using an ordinal protein expression technique. For example, first, a vector including a gene sequence coding for the recombinant protein of the present disclosure is prepared. An example of the vector is a plasmid. Then, cells (e.g., Escherichia coli) are transformed with this vector. These cells are incubated to produce the recombinant protein of the present disclosure.

In order to obtain the scFv antibody fragment efficiently, it is beneficial that the recombinant protein of the present disclosure is produced by a refolding method. Non Patent Literature 3 discloses the refolding method.

Non Patent Literature 3

Jun Kamishikiryo et. al., “Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)”, THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 286, NO. 27, p.p. 23823-23830.

Examples of the technique of the present disclosure are as follows.

1st aspect: A recombinant protein which binds specifically to troponin I derived from human myocardium. The recombinant protein includes a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 63, and a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 65.

2nd aspect: In the recombinant protein according to the 1st aspect, the recombinant protein may be an antibody.

3rd aspect: In the recombinant protein according to the 1st aspect, the recombinant protein may be an antibody fragment.

4th aspect: In the recombinant protein according to the 3rd aspect, the antibody fragment may be a Fab antibody fragment.

5th aspect: In the recombinant protein according to the 3rd aspect, the antibody fragment may be a F(ab′)₂antibody fragment.

6th aspect: In the recombinant protein according to the 3rd aspect, the antibody fragment may be an scFv antibody fragment.

7th aspect: A method for binding a recombinant protein specifically to troponin I derived from human myocardium, includes the following steps. A step (a) is a step of preparing the recombinant protein. The recombinant protein includes a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63, and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65. A step (b) is a step of bringing the recombinant protein into contact with the troponin I derived from human myocardium to bind the recombinant protein specifically to the troponin I derived from human myocardium. In this aspect, for example, the step (b) may be carried out in vitro.

8th aspect: In the method according to the 7th aspect, the recombinant protein may be an antibody.

9th aspect: In the method according to the 7th aspect, the recombinant protein may be an antibody fragment.

10th aspect: In the method according to the 9th aspect, the antibody fragment may be a Fab antibody fragment.

11th aspect: In the method according to the 9th aspect, the antibody fragment may be a F(ab′)₂antibody fragment.

12th aspect: In the method according to the 9th aspect, the antibody fragment may be scFv antibody fragment.

EXAMPLES

An example for supporting an exemplary embodiment of the present disclosure is described below.

Example 1

Table 1, Table 2, Table 3, and Table 4 show the primers used in Example 1.

Table 1 shows the forward mixture primers (primers 1-21, SEQ ID NOS: 02-22) for amplifying a light chain variable region.

Table 2 shows the forward mixture primers (primers 22-44, SEQ ID NOS: 23-45) for amplifying a heavy chain variable region.

Table 3 shows the reverse mixture primers (primers 45-49, SEQ ID NOS: 46-50) for amplifying a light chain variable region.

Table 4 shows the reverse mixture primers (primers 50-55, SEQ ID NOS: 51-56) for amplifying a heavy chain variable region.

TABLE 1

Primer 1	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	02	TWCTCWCCCARTC

Primer
2	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTS
	03	TGMTSACYCAGTC

Primer
3	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	04	TGMTMACTCAGTC

Primer 4	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	05	TGHTRWCACAGTC

Primer
5	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	06	TRATGACMCAGTC

Primer 6	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTM
	07	AGATRAMCCAGTC

Primer 7	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTC
	08	AGATGAYDCAGTC

Primer 8	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTT
	09	TGCTGACTCAGTC

Primer 9	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	10	TTCTCAWCCAGTC

Primer 10	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	11	WGCTSACCCAATC

Primer 11	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTS
	12	TRATGACCCARTC

Primer 12	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYRTTK
	13	TGATGACCCAVAC

Primer 13	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATYC
	14	AGATGACACAGAC

Primer 14	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	15	TGATGACACAACC

Primer 15	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATCC
	16	AGCTGACTCAGCC

Primer 16	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	17	TGATGACBCAGKC

Primer 17	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	18	TGATAACYCAGGA

Primer 18	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	19	TGATGACCCAGWT

Primer 19	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYGTGS
	20	TGMTSACYCAGTC

Primer 20	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYGCTG
	21	TTGTACTCAGGAATC

Primer
21	SEQ ID NO:	CCTTTCTATGCGGCCCAGCCGGCCATGGCCGAYATTG
	22	TDHTVWCHCAGTC

TABLE 2

Primer 22	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	23	AKGTRMAGCTTCAGGAGYC

Primer
23	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	24	AGGTNCAGCTBCAGCAGTC

Primer
24	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	25	AGGTGCAGCTGAAGSASTC

Primer 25	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	26	AGSTBCAGCTGCAGCAGTC

Primer 26	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	27	AGGTYCAGCTYCAGCAGTC

Primer 27	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	28	ARGTCCARCTGCAACARTC

Primer 28	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	29	AGGTYCAGCTBCAGCARTC

Primer 29	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	30	AGGTYCARCTKCAGCAGTC

Primer 30	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	31	AGGTCCACGTGAAGCAGTC

Primer
31	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	32	AGGTGAASSTGGTGGARTC

Primer
32	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	33	AVGTGAWGYTGGTGGAGTC

Primer 33	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	34	AGGTGAAGGTCATCGAGTC

Primer 34	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCS
	35	AGGTGCAGSKGGTGGAGTC

Primer 35	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	36	AKGTGCAMCTGGTGGAGTC

Primer 36	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	37	AAGTGCAVCTGGTGGAGTC

Primer 37	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	38	AGGTGAAGCTGATGGARTC

Primer 38	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	39	AGGTGCARCTTGTTGAGTC

Primer 39	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	40	ARGTRAAGCTTCTCGAGTC

Primer 40	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	41	AAGTGAARSTTGAGGAGTC

Primer 41	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	42	AAGTGATGCTGGTGGAGTC

Primer 42	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	43	AGGTTACTCTRAAAGWGTSTG

Primer 43	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCC
	44	AGGTCCAAYTVCAGCARCC

Primer 44	SEQ ID NO:	AGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCG
	45	ATGTGAACTTGGAAGTGTC

TABLE 3

Primer 45	SEQ ID NO:	ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC
	46	CGTTTGATTTCCARCTTKG

Primer 46	SEQ ID NO:	ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC
	47	CGTTTTATTTCCAGCTTGG

Primer 47	SEQ ID NO:	ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC
	48	CGTTTSAGCTCCAGCTTGG

Primer 48	SEQ ID NO:	ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC
	49	CGTTYWATTTCCAACTTWG

Primer 49	SEQ ID NO:	ACCAGAGCCGCCGCCGCCGCTACCACCACCACCC
	50	CCTAGGACAGTCAGTTTGG

TABLE 4

Primer 50	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGAGGAAACGGTGAC
	NO: 51	CGTGGT

Primer 51	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGAGGAGACTGTGAG
	NO: 52	AGTGGT

Primer 52	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGAGGAGACGGTGAC
	NO: 53	TGAGRT

Primer 53	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGAGGAAGACTGTAG
	NO: 54	AGTGGT

Primer 54	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGCGGAGACASTGAC
	NO: 55	CAGAGT

Primer 55	SEQ ID	CGGCACCGGCGCACCTGCGGCCGCYGCAGAGACASTGAC
	NO: 56	CAGAGT

Step (a-1) Preparation of a Hybridoma (Derived from Mouse Spleen) Capable of Producing Monoclonal Antibodies which Specifically Bind to Troponin I Derived from Human Myocardium

A peptide having an amino acid sequence (SEQ ID NO: 01, purchased from Sigma Aldrich Japan Co., Ltd.,

RPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAK) contained in troponin I derived from human myocardium was connected to human serum albumin (purchased from Sigma Aldrich Japan Co. Ltd.) using a sulfo-SMCC cross linker (purchased from Servo Fischer Scientific Co., Ltd.).

More particularly, the sulfo-SMCC cross linker (0.5 mg) was dissolved in 100 microliters of a phosphate buffered saline so as to obtain a first aqueous solution. This first aqueous solution was left under a temperature of 50 degrees Celsius for ten minutes.

The human serum albumin (10 mg) was dissolved in one milliliter of a phosphate buffered saline to obtain a second aqueous solution.

The first aqueous solution was mixed with the second aqueous solution to obtain a mixture. The mixture was left at rest for 30 minutes. In this way, the sulfo-SMCC cross linker was connected to the human serum albumin.

The mixture was passed through a column (purchased from GE health care, trade name: PD10) to remove the unreacted sulfo-SMCC cross linker.

The above-mentioned peptide (SEQ ID NO: 01, 1.5 mg) was dissolved in dimethylsulfoxide (hereinafter, referred to as “DMSO”) to obtain a DMSO solution. The DMSO solution (100 microliters) was added to the mixture (1 mL) having a concentration of 2 mg/ml. Afterwards, the mixture was left overnight to connect the sulfo-SMCC cross linker to the peptide (SEQ ID NO: 01).

In this way, human serum albumin modified with the peptide having the amino acid sequence (SEQ ID NO: 01) contained in the troponin I was obtained. Hereinafter, this human serum albumin is referred to as “troponin-modified HSA”.

A complete Freud adjuvant (purchased from Wako Pure Chemical Industries Co., Ltd.) and the troponin-modified HSA were mixed to obtain a mixture. This mixture was injected to a BALB/c mouse. The BALB/c mouse is a kind of albino mouse.

Two weeks later, a mixture of phosphate buffered saline (hereinafter, referred to as “PBS”) and troponin-modified HSA was injected into the BALB/c mouse. This was repeated once again. In this way, the BALB/c mouse was immunized by troponin-modified HSA for one month. In other words, by administering the mixture to the BALB/c mouse, antibodies against troponin-modified HSA were produced in the body of the BALB/c mouse.

The spleen of the immunized BALB/c mouse was taken out. In accordance with the cell fusion method disclosed in Non Patent Literature 4, hybridomas were obtained. Afterwards, the hybridomas were incubated in a culture fluid. The number of hybridomas (cells) after the incubation was approximately 5×10⁶. The hybridomas obtained in this way were capable of producing the monoclonal antibody which specifically bound to troponin I derived from human myocardium.

Non Patent Literature 4

G. Kohler et al., Nature, 256, 495 (1975)

Step (a-2) Extraction of Total Mouse RNAs from the Hybridoma Cells

In order to destroy the cell membrane of the cultured hybridomas, one milliliter of TRIzol (Purchased from Invitrogen Co., Ltd.) was added to the culture fluid containing the hybridomas, and the culture fluid was stirred well.

Then, a chloroform liquid having a volume of 0.2 mL (degree of purity: 99.9%) was added to the culture fluid, and the culture fluid was stirred well again.

The culture fluid was subjected to a centrifugal separation at an acceleration of gravity of 117600 m/s²under a temperature of 4 degrees Celsius for 15 minutes. The supernatant (500 μL) was acquired. Isopropanol (500 μL) was added to the obtained supernatant and left at rest under room temperature for ten minutes.

The culture fluid was again subject to a centrifugal separation having a condition identical to the above-mentioned condition to obtain a precipitate. A seventy-five percent ethanol aqueous solution (1 mL) was added to the obtained precipitate so as to obtain an ethanol solution.

The ethanol solution was subjected to a centrifugal separation at an acceleration of gravity of 73500 m/s²for five minutes. The precipitate was dried. In this way, total mouse RNAs were obtained.

Step (b-1) Extraction of mRNA from the Total Mouse RNAs

Using an Oligotex™-dT30 <Super> mRNA Purification kit (purchased from Takara bio Co., Ltd.), mRNA was extracted from the total mouse RNAs obtained in the step (a-2).

RNase-free water (100 μL) was injected into a microtube. This microtube was set at a block incubator (purchased from ASTEC CO. LTD.) and heated under a temperature of 70 degrees Celsius for one hour.

The total mouse RNAs were dissolved in the RNase-free water (100 μL).

A 2× binding buffered solution (100 μL) included in the kit and an oligotex (10 μL) included in the kit were mixed with the RNase-free water (100 μL). Subsequently, the mixture was left at rest under a temperature of 70 degrees Celsius for three minutes. Furthermore, the mixture was left at rest under room temperature for ten minutes.

The mixture was subjected to a centrifugal separation at an acceleration of gravity of 147000 m/s²for five minutes. The supernatant was removed, and the precipitate was suspended in Wash buffer (350 μL) included in the kit. The suspension liquid was supplied to a column included in the kit. The column was subjected to a centrifugal separation at an acceleration of gravity of 147000 m/s²for 30 seconds.

The Wash buffer (350 μL) was supplied to the column to wash the column. The column was subjected to a centrifugal separation at an acceleration of gravity of 147000 m/s²again for 30 seconds.

A microtube for sample collection was attached to the bottom of the column.

In order to extract mRNA contained in the column, RNase-free water (20 μL) contained in the microtube was supplied to the column. Subsequently, the column was subjected to a centrifugal separation at an acceleration of gravity of 147000 m/s²for three minutes. Again, RNase-free water (20 μL) was supplied to the column, and the column was subjected to a centrifugal separation at an acceleration of gravity of 147000 m/s²for three minutes.

Thus, the extract liquid containing the mRNA was obtained in the microtube.

(Step b-2) Reverse-Transcription from mRNA to cDNA

The mRNA contained in the obtained extract liquid was reverse-transcripted with a reverse-transcriptase (purchased from Takara bio Co., Ltd, trade name: Primersript) to obtain a solution containing cDNA.

Step (b-3-1) Amplification of the Gene Coding for the Light Chain Variable Region Using the cDNA

The gene fragment (SEQ ID NO: 58, hereinafter, referred to as “VL gene fragment”) coding for the light chain variable region of the above-mentioned monoclonal antibody was amplified by a PCR method using the cDNA contained in the solution, the forward primers 1-21 (SEQ ID NOS: 02-22), and the reverse primers 1-5 (SEQ ID NOS: 23-27). The polymerase used in this PCR method was purchased from Takara bio Co., Ltd under a trade name of TaKaRa Ex Taq Hot start Version.

The protocol of this PCR method is shown in Table 5.

	TABLE 5

	One cycle	ninety six degrees Celsius for thirty seconds
		fifty two degrees Celsius for one minute
		sixty eight degrees Celsius for one minute

The number of the cycles: 35.

Finally, the solution was left at 68 degrees Celsius for four minutes. In this way, a PCR solution was obtained. This PCR solution contained the amplified VL gene fragment (SEQ ID NO: 58).

For the confirmation and purification of the amplified VL gene fragment, the obtained PCR solution was subjected to an electrophoresis using a gel containing agarose having a concentration of 2% by weight.

Step (b-3-2) Amplification of the Gene Coding for the Heavy Chain Variable Region Using the cDNA

The gene fragment (SEQ ID NO: 57, hereinafter, referred to as “VH gene fragment”) coding for the heavy chain variable region of the above-mentioned monoclonal antibody was amplified by a PCR method using the cDNA contained in the solution, the forward primers 22-44 (SEQ ID NOS: 28-50), and the reverse primers 6-11 (SEQ ID NOS: 51-56). The polymerase used in this PCR method was purchased from Takara bio Co., Ltd under a trade name of TaKaRa Ex Tag Hot start Version.

The protocol of this PCR method was identical to that used for the VL gene fragment.

Finally, the solution was left at 68 degrees Celsius for four minutes. In this way, a PCR solution was obtained. This PCR solution contained the amplified VH gene fragment (SEQ ID NO: 57).

For the confirmation of the generation of the VH gene fragment and for the purification of the VH gene fragment, the obtained PCR solution was subjected to an electrophoresis using a gel containing agarose having a concentration of 2% by weight.

Step (b-4) Connection of the VL Gene Fragment and the VH Gene Fragment

The purified VH gene fragment (SEQ ID NO: 57) was connected to the purified VL gene fragment (SEQ ID NO: 58) using an overlap extension PCR method. In this way, the gene fragment (SEQ ID NO: 59, hereinafter, referred to as “scFv gene fragment”) coding for the scFv antibody fragment of the above-mentioned monoclonal antibody was obtained. The obtained gene fragment (SEQ ID NO: 59) was modified with restriction enzyme sites Nco1 and Not 1 at the 5′-end and 3′-end thereof, respectively.

Step (c-1) Introduction of the Gene to a Vector

The scFv gene fragment was ligated into a protein expression vector (purchased from Takara bio Co., Ltd, trade name: pET22b(+)). The detail of the ligation is described below.

First, the scFv gene fragment was treated with restriction enzymes Nco1 and Not1 (both of which were purchased from Takara bio Co., Ltd.). The scFv gene fragment was purified by an electrophoresis method to obtain an aqueous solution containing the scFv gene fragment.

The protein expression vector was also treated with restriction enzymes Nco1 and Not1 (both of which were purchased from Takara bio Co., Ltd.). The protein expression vector was also purified by an electrophoresis method to obtain an aqueous solution containing the protein expression vector.

These two aqueous solutions were mixed to obtain a mixture.

An enzyme (purchased from Toyobo Co., Ltd., trade name: Ligation High ver. 2) was added to the mixture, and the mixture was left under a temperature of 16 degrees Celsius for two hours. In this way, the scFv gene fragment was ligated into the protein expression vector.

Escherichia coli cells (purchased from Takara bio Co., Ltd., trade name; DH5α competent cell) were transformed with the protein expression vector in which the scFv gene fragment was thus ligated.

Subsequently, the Escherichia coli cells were incubated for sixteen hours on an LB plate culture medium containing ampicillin having a concentration of 100 μg/mL. After the incubation, a single colony formed on the LB plate culture medium was picked up. The single colony was supplied to an LB liquid culture medium (5 mL) containing ampicillin having a concentration of 100 μg/mL, and the colony was incubated for 16 hours.

In order to remove an unnecessary gene sequence included in the protein expression vector pET22b(+), the protein expression vector pET22b(+) was extracted from this LB liquid culture medium using a kit (QIAGEN Co., Ltd. trade name: QIAprep spin miniprep kit). By a PCR method using the extracted protein expression vector pET22b(+), the primer 56 (SEQ ID NO: 67), and the primer 57 (SEQ ID NO: 68), the signal sequence (DNA sequence, SEQ ID NO: 60) of the protein expression vector pET22b(+) was removed. Thus, the expression vector coding for the wild type scFv antibody fragment was obtained.

Step (c-2) Introduction of the Mutations to the Vector

The expression vector obtained in the step (c-1) included the scFv gene fragment (SEQ ID NO: 59). Among the 729 bases constituting the scFv gene fragment (SEQ ID NO: 59) included in the expression vector, seven bases were substituted. In more detail, the 589th adenine (A), the 590th cytosine (C), the 607th thymine (T), the 608th cytosine (C), the 609th cytosine (C), the 613th adenine (A), and the 615th cytosine (C) were substituted with guanine (G), adenine (A), guanine (G), adenine (A), thymine (T), guanine (G), and thymine (T), respectively.

More particularly, as shown in FIG. 2, a PCR method using the primer 58 (SEQ ID NO: 69), the primer 59 (SEQ ID NO: 70) and the expression vector obtained in the step (c-1) was performed. The primer 58 (SEQ ID NO: 69) was complementary to the gene sequence from 579^thbase to 626^thbase included in the scFv gene fragment (SEQ ID NO: 59) except for the seven bases to be substituted. The primer 59 (SEQ ID NO: 70) was complementary to the gene sequence from 579^thbase to 626^thbase included in the gene fragment complimentary to the scFv gene fragment (SEQ ID NO: 59) except for the seven bases to be substituted. The PCR method shown in FIG. 2 allowed the seven bases (AC, TCC, A, C) included in the expression vector coding for the wild type scFv antibody fragment to be substituted with the different seven bases (GA, GAT, G, T). Thus, the expression vector containing the gene sequence (SEQ ID NO: 71) encoding the mutant scFv was obtained.

Step (c-3) Acquisition of the Protein Using the Vector

Escherichia coli cells (purchased from Takara bio Co., Ltd, trade name: BL21(DE3)) were transformed with the vector obtained in the step (c-2). Subsequently, the Escherichia coli cells were incubated on an LB plate culture medium containing ampicillin having a concentration of 100 μg/mL under a temperature of 37 degrees Celsius for 16 hours.

After the incubation, a single colony formed on the LB plate culture medium was picked up. The single colony was supplied to an LB liquid culture medium containing ampicillin (500 mL) having a concentration of 100 μg/mL. Subsequently, the Escherichia coli cells contained in the single colony were propagated in such a manner that the absorbance of the LB liquid culture medium at a wavelength of 600 nanometers was adjusted to 0.5.

Furthermore, an aqueous solution of isopropyl beta-D-thiogalactopyranoside (0.5 mL) having a concentration of 1 M was added to the LB liquid culture medium. Afterwards, the Escherichia coli cells were incubated with shaking under a temperature of 37 degrees Celsius for five hours. In this way, a culture fluid was obtained.

The obtained culture fluid was subjected to a centrifugal separation at an acceleration of gravity of 49000 m/s²under a temperature of 4 degrees Celsius for five minutes. The precipitation containing the Escherichia coli cells was again suspended in a phosphate buffered saline (50 mL).

The suspension was subjected to an ultrasonic treatment to crush the Escherichia coli cells. The solution containing the crushed Escherichia coli cells was subjected to a centrifugal separation at an acceleration of gravity of 98000 m/s²under a temperature of 4 degrees Celsius for thirty minutes. In this way, the precipitation was obtained.

The precipitation was washed twice with a phosphate buffered saline containing a surface active agent (purchased from Wako Pure Chemical Industries Co., Ltd., trade name: Triton X-100) having a concentration of 4%. The precipitation was further washed with a phosphate buffered saline without containing a surface active agent.

An aqueous solution A (10 mL) containing chemical reagents shown in Table 6 was added to the precipitation.

	TABLE 6

	Chemical reagents	Concentration

	Guanidine hydrochloride	6M
	Sodium chloride	0.1M
	MES buffer solution	50 mM
	Ethylene diamine tetraacetic acid	10 mM

The aqueous solution A had a pH of 6.

Subsequently, the aqueous solution A was left under a temperature of 4 degrees Celsius for eighteen hours. In this way, the precipitation was dissolved.

The aqueous solution A was passed through a filter (purchased from Sartorius, trade name: Minisart) having a mesh size of 0.45 μm to remove the residue. In this way, the filtrate was obtained.

Two milliliters of an aqueous solution B was added dropwise to the filtrate (1 mL). The composition of the aqueous solution B (concentrations of chemical reagents contained in the aqueous solution B) is shown in Table 7.

	TABLE 7

	Chemical reagents	Concentration

	Tris-HCl	0.1M
	Ethylene diamine tetraacetic acid	2 mM
	Arginine hydrochloride	1.0M
	Cystamine	3.73 mM
	Cysteamine hydrochloride	6.73 mM

The aqueous solution B had a pH of 8.0. In this way, an aqueous solution having a volume of 3 mL was obtained.

The aqueous solution (3 mL) was added dropwise to an aqueous solution having a volume of one liter which contained the chemical reagents shown in Table 7. Afterwards, the obtained aqueous solution was stirred under a temperature of 4 degrees Celsius for 96 hours. In this way, the mutant scFv antibody fragment (SEQ ID NO: 61) was obtained.

Subsequently, the solution was concentrated using a filtration unit (purchased from Sartorius, trade name: VIVAFLOW50) so that the solution had a volume of 10 milliliters. The mutant scFv antibody fragment contained in the solution was purified with a column (purchased from GE healthcare, trade name: HiLoad 26/60 Superdex 75 pg).

The detail of the amino acid sequence (SEQ ID NO: 61) of the mutant scFv antibody fragment is described below.

Light chain variable region (SEQ ID NO: 63):
DVVLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLV

SKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLEIKR

The amino acid sequence modified at the N-terminal of the light chain variable region: None

The amino acid sequence modified at the C-terminal of the light chain variable region: None

Linker (SEQ ID NO: 64):

GGGGSGGGGSGGGGS

Heavy chain variable region (SEQ ID NO: 65):

QLQLQQSGAELVRSGASVKLSCTASGFNIKDYYMNWVKQRPEQGLE

WIGWIDPANGDTAYAPRFQVKADMTADKDSDTAYLQLSSLTSEDTAVYYC

NADLPMDQWGQGTSVTVSS

The amino acid sequence modified at the N-terminal of the heavy chain variable region: None

The amino acid sequence modified at the C-terminal of the heavy chain variable region:

AAALEHHHHHH

(SEQ ID NO: 66)

Step (d) Calculation of Association Rate Constant and Dissociation Rate Constant

Using an intermolecular interaction analyzer Biacore T100 (purchased from GE health care company), the association rate constant and the dissociation rate constant of the mutant scFv antibody fragment were calculated in accordance with the manual attached to the intermolecular interaction analyzer Biacore T100.

Troponin I (purchased from Funakoshi) derived from human myocardium having approximately 500 RU (Resonance Unit) was fixed on a CM5 chip (purchased from GE health care company). This CM5 chip was set in the Biacore T100. Then, aqueous solutions (concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM; volume: 150 microliters) containing the mutant scFv antibody fragment were flowed through the Biacore T100. The association rate constant and the dissociation rate constant measured with the intermolecular interaction analyzer Biacore T100 are shown in Table 9.

Comparative Example 1

In Comparative Example 1, the experiment similar to Example 1 was conducted except that the step (c-2) was not conducted. In this way, the wild type scFv antibody fragment consisting of the amino acid sequence represented by SEQ ID NO: 62 was obtained. Similarly to Example 1, the association rate constant and the dissociation rate constant of the wild-type scFv antibody fragment were measured. The results are shown in Table 9.

The differences between the wild type scFv antibody fragment (SEQ ID NO: 62) and the mutant scFv antibody fragment (SEQ ID NO: 61) are shown in Table 8.

	TABLE 8

	Wild type scFv	Mutant scFv
	antibody fragment	antibody fragment
	(SEQ ID: 62)	(SEQ ID: 61)

197^thamino acid in the scFv	T	D
antibody fragment
203^rdamino acid in the scFv	S	D
antibody fragment
205^thamino acid in the scFv	N	D
antibody fragment

	TABLE 9

	Comparative
	example 1	Example 1

Association rate constant	5.173 × 10⁺⁴	1.24 × 10⁺⁵
Dissociation rate constant	1.361 × 10⁻⁴	2.14 × 10⁻⁴

As is clear from Table 9, the mutant scFv antibody fragment according to Example 1 has a higher association rate constant than that of the wild-type scFv antibody fragment according to Comparative Example 1. This means that the mutant scFv antibody fragment bound specifically to troponin I derived from human myocardium more quickly than the wild-type scFv antibody fragment.

Industrial Applicability

The recombinant protein and the method according to the present disclosure can be used for a sensor for detecting acute myocardial infarction.

Claims

What is claimed is:

1. A recombinant protein which binds specifically to troponin I derived from human myocardium, the recombinant protein comprising:

a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 63; and

a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65.

2. The recombinant protein according to claim 1, wherein the recombinant protein is an antibody.

3. The recombinant protein according to claim 1, wherein the recombinant protein is an antibody fragment.

4. The recombinant protein according to claim 3, wherein the antibody fragment is a Fab antibody fragment.

5. The recombinant protein according to claim 3, wherein the antibody fragment is a F(ab′)₂antibody fragment.

6. The recombinant protein according to claim 3, wherein the antibody fragment is an scFv antibody fragment.

7. A method for binding a recombinant protein specifically to troponin I derived from human myocardium, the method comprising:

a step (a) of preparing the recombinant protein, wherein

the recombinant protein comprises:

a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 65; and

a step (b) of bringing the recombinant protein into contact with the troponin I derived from human myocardium to bind the recombinant protein specifically to the troponin I derived from human myocardium.

8. The method according to claim 7, wherein the recombinant protein is an antibody.

9. The method according to claim 7, wherein the recombinant protein is an antibody fragment.

10. The method according to claim 9, wherein the antibody fragment is a Fab antibody fragment.

11. The method according to claim 9, wherein the antibody fragment is a F(ab′)₂antibody fragment.

12. The method according to claim 9, wherein the antibody fragment is an scFv antibody fragment.